Abstract LB154: Circ0003039 interacts with calreticulin to inhibit the progression of hepatocellular carcinoma by suppressing wnt/beta-catenin signaling pathway

Abstract Background: Circular RNAs (circRNAs) are covalently closed and single-stranded RNAs which play critical roles in various biological processes and diseases including cancers. The functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) are still need to be clarified. Methods: Custom circRNA microarray was performed to investigate the profile of circRNAs in HCC. Expression of circ0003039 and its clinical significance in 60 pairs of HCC and matched non-cancer liver tissues were identified and confirmed by microarray and quantitative RT-PCR. The biological functions of circ0003039 were investigated by gain-and loss-of-function experiments in vitro and in vivo. RNA pull-down, mass spectrometry, RNA-immunoprecipitation, Co-immunoprecipitation, fluorescent in situ hybridization were used to identify circ0003039-protein interaction. The downstream pathway regulated by circ0003039 was explored using RNA-seq and verified using western blot and immunofluorescence. Results: Expression of circ0003039 was reduced in HCC tissues and positively associated with the overall survival of HCC patients. Circ0003039 was located in cytoplasm of HCC cells. Ectopic overexpression of circ0003039 inhibited the proliferation, colony formation, migration and invasion of HCC cells and the growth of HCC xenograft in mice, while knockdown of circ0003039 had opposite effects in HCC cells. Mechanistically, circ0003039 bound to the N-terminal domain of CALR protein and acted as a scaffold to enhance the interaction of CALR with CAPN2, which promoted the degradation of CALR protein by the enzymatic activity of CAPN2. Rescue experiments showed that circ0003039 inhibited HCC proliferation via suppressing CALR protein. Finally, we found that circ0003039 inhibited HCC cells by suppressing CALR-mediated wnt-βcatenin signaling pathway. Conclusions: Our study demonstrates that circ0003039 bind to N-terminal domain of CALR and facilitated CAPN2 to degrade CALR protein, which inhibits the development and progression of HCC via suppressing CALR-mediated wnt-βcatenin signaling pathway, and circ0003039 is a potential prognostic biomarker and therapeutic target for HCC. Citation Format: Yue-Ning Wang, Yu-Feng Zhou, Ji Liu, Mei-Yin Zhang, Shuo-Cheng Wang, Shi-Juan Mai, Hui-Yun Wang. Circ0003039 interacts with calreticulin to inhibit the progression of hepatocellular carcinoma by suppressing wnt/beta-catenin signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB154.